Ashwin Amurthur, Peter Qiao, Michael Magaraci, Avin Veerakumar How - - PowerPoint PPT Presentation

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Ashwin Amurthur, Peter Qiao, Michael Magaraci, Avin Veerakumar How - - PowerPoint PPT Presentation

Aug 22, 2022 458 likes •949 views

Ashwin Amurthur, Peter Qiao, Michael Magaraci, Avin Veerakumar How can Synthetic Biology Improve Medicine? Current Medical Therapies Specific Effects Non-specific effects Parameters: 1. Targeting Specificity 2. Dosage Control

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SLIDE 1

Ashwin Amurthur, Peter Qiao, Michael Magaraci, Avin Veerakumar

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SLIDE 2

How can Synthetic Biology Improve Medicine?

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SLIDE 3

Current Medical Therapies

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

Specific Effects Non-specific effects Parameters: 1. Targeting Specificity 2. Dosage Control

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SLIDE 4

How can we use Synthetic Biology to improve Targeting Specificity and Dose Control? By Engineering Bacteria to act as the Therapeutic

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SLIDE 5

Passive Diffusion Dose Scheduling Spatial – Radiation Therapy

  • r

Cellular – mAb, Chemotherapy

Cancer As a Case Study

Current Cancer Therapies

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

Dose Control Targeting Specificity

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SLIDE 6

Passive Diffusion Dose Scheduling Spatial – Radiation Therapy

  • r

Cellular – mAb, Chemotherapy

Cancer As a Case Study

Current Cancer Therapies Proposed Bacterial Therapy

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

Dose Control Targeting Specificity Dose Control Targeting Specificity Combined Spatial and Cellular Targeting Active Diffusion Tunable Transgene Expression System

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SLIDE 7
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SLIDE 8

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

Proposed System Video

Credit to Autodesk for providing us with Maya!

Tumor Cell Cytoplasm HER2 Cell Membrane

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SLIDE 9
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SLIDE 10

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

Achieving Light-Activated Cell Lysis

Goal is to demonstrate that:

  • YF1/FixJ (pDawn) blue light sensor can be activated for

downstream transgene expression

  • YF1/FixJ blue light sensor allows for light dependent lysis
  • f mammalian cells
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SLIDE 11

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

YF1/FixJ BL Sensor Allows For Light- Dependent Transgene Expression

0h 1h 2h 3h 4h 6h 8h 22h

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Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

Cytolysin A (ClyA)

(Wallace et. al, 2000)

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Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

Light-triggered lysis of mammalian cells by pDawn-ClyA bacteria

(D) (L) pDawn-His-ClyA Dark pDawn-His-ClyA Light

pDawn-mCherry Dark pDawn-mCherry Light

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SLIDE 14

Spatial control of cell lysis

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

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SLIDE 15

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

Characterizing ClyA-mediated lysis

  • f SKBR3 cancer cells

ClyA Cytotoxicity in SKBR3 cells (High HER2 Level Expression) ClyA Cytotoxicity in HEK293T cells (Basal HER2 Level Expression) **** p<0.0001

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SLIDE 16

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

Targeting Cancer Cells

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Ice Nucleation Protein, NC Domain

  • Outer membrane protein

Discovered in P. syringae

  • Promotes ice crystallization
  • Can remove internal repeats

and display proteins on the surface of E. coli

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

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SLIDE 18

Displaying DARPin H10-2-G3

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

DARPin H10-2-G3

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Assaying Display of DARPin H10-2-G3

  • HA tag allowed detection of surface proteins

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

INPNC-HA (+IPTG) INPNC-HA (-IPTG)

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SLIDE 20

DARPin was Successfully Displayed!

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

DARPin-HA (-IPTG) DARPin-HA (+IPTG)

INPNC-DARPin-HA (-IPTG) INPNC-DARPin-HA (+IPTG)

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SLIDE 21

Can our Bacteria Bind to Cancer Cells?

  • SKBR3 Cells are derived

from breast tumors

  • Overexpress HER2

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

DAPI, HER2

Breast Tumor Tissue1 Our Cultured SKBR3 Cells

  • 1. Y. Xiao, X. Gao, G. Gannot, M.R. Emmert-Buck, S. Srivastava,

P.D. Wagner, M.A.Amos and P.E. Barker. Quantitation of HER2 and telomerase biomarkers in solid tumors with IgY antibodies and nanocrystal detection. International Journal of Cancer

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DARPin-displaying Bacteria Bind to SKBR3 Cells Preferentially

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

HEK293T (Basal HER2) SKBR3 (High HER2)

DAPI HER2 eGFP

+E. coli (+IPTG) +E. coli (-IPTG)

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DARPin-displaying Bacteria Bind to SKBR3 Cells Preferentially

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

DAPI, HER2, eGFP

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Submitted BioBricks

  • ClyA BioBricks: BBa_K811000-

K811002

  • INPNC BioBricks: BBa_K811003-

K811004

  • INPNC-MCS: BBa_K811005
  • General surface display vector

for any iGEM team

  • Only one ligation needed!
  • Regional “Best BioBrick,

Engineered”

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

INPNC-MCS Platform

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SLIDE 25

Human Practices

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SLIDE 26
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VerifiGEM User Interface

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SLIDE 28
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SLIDE 29 http://2009.igem.org/Team:Stanford
  • Many people have tried

to apply synthetic biology to treat disease. – Over 75 Health/Medicine teams since 2009 – Many, many papers since 1995!

  • Where are they now?
  • Why?

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

Looking Back on Eight Years of iGEM: Where are We Now?

http://2008.igem.org/Team:MIT http://2011.igem.org/Team:Missouri_Miners
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SLIDE 30

Perception Barriers to Bacterial Therapeutics

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

Perception Barriers

  • Negative portrayal
  • f E. coli in the

media

  • Public unfamiliar

with synthetic biology

Public perception of E. coli is negative

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SLIDE 31

Perception Barriers: The Hype Cycle

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion Linden, A. (2003). Understanding gartner’s hype cycles. Conshohocken: Gartner.

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SLIDE 32

Education & Outreach

  • Presentation/Q&A

session with high school students

  • Clark Park Science

Discovery Day

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

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Biological Barriers to Bacterial Therapeutics

Biological Barriers

  • E. coli produces

compounds that are immunogenic

  • Lab strains are poor

candidates for use in the human body

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

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SLIDE 34

Addressing the Biological Barrier

  • E. coli Nissle 1917

– Nonpathogenic – Used as a probiotic supplement in Canada and Europe – Low immunogenicity

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

http://www.npr.org/blogs/thesalt/2012/07/09/156381323/confusion-at-the-yogurt-aisle-time-for- probiotics-101 http://www.msnbc.msn.com/id/27454348/ns/health- diet_and_nutrition/t/probiotics-more-hype- help/#.UHd2ScXA-f4 http://sitemaker.umich.edu/huffnagl e.lab/_the_probiotics_revolution___ book_
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The pDawn Expression System is Functional in Nissle 1917

  • Chemically

competent Nissle 1917 achieved light controlled ClyA hemolysis

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

+1000 μg pDawn-his-ClyA +H2O (Neg. Control)

pDawn-mCherry Light pDawn-mCherry Dark pDawn-His-clyA Dark pDawn-His-clyA Light

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SLIDE 36

Recommendation for Future H&M iGEM Teams

  • When designing a project, keep clinical

endpoints in mind

  • Utilize strains of bacteria like Nissle 1917

– Scientific benefits – Perception benefits

  • Use outreach events as an opportunity to

smooth out the hype curve

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

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Accomplishments

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

 Functionalized bacteria as a light- activated drug delivery platform  Demonstrated light-dependent hemolysis in a spatially controlled manner Light-Activated Drug Delivery Surface Display and Targeting  Created an easily adapted surface display BioBrick  First to display DARPin on the surface of E. coli  Showed HER-2 dependent binding

  • f bacteria to human cells

 VerifiGEM  Analyzed barriers to bacterial therapeutics  Nissle 1917 – future chassis for bacterial therapeutics Human Practices A Novel, Modular Platform for a Targeted Bacterial Therapeutic

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SLIDE 38

Future Directions

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

 Use other wavelengths of light (such as red) with more clinical relevance  Decrease non-specific binding of our engineered therapeutic through modification of E. coli surface Optimization of our Platform  Port the entire system into Nissle 1917  Test our system in a mouse model in vitro Increase Therapeutic Viability

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SLIDE 39

Acknowledgements

Introduction Light-Based Drug Delivery Surface Display & Targeting Human Practices Conclusion

  • Dr. Casim A. Sarkar
  • Dr. Jordan Miller
  • Dr. Mark Goulian
  • Najaf Shah
  • Daphne Ng
  • Sevile Mannickarottu
  • Henry Ma
  • Autodesk
  • Nikita Singh
  • Karsticum Computing

Inc.

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Sponsors

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Questions and Answers

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Supplementary Slides

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Testing the System with mCherry

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Testing the System with mCherry

  • Fused INPNC to mCherry

with 12aa GS Linker

  • Sonicated INPNC-

mCherry expressing E. coli and separated lysate and membrane fraction

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DARPin Binding

HEK293T + 3nM DARPin SKBR3 + 3nM DARPin

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Coomassie Gel

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Selective binding of SKBR3 cells in HEK293T/SKBR3 Co-Culture

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SLIDE 48

Light-Activated Cytotoxicity